Many molecules that direct the establishment of polarities within epithelial sheets have been identified. However, there is a critical gap in our understanding of how these proteins become localized to specific places within the cell. With this in mind, the objective of this application is to establish a set of tools and techniques to address the dynamic localization of epithelial polarity proteins. To date, most polarity proteins in Drosophila have been examined under steady-state conditions in fixed tissue preparations. The focus of the proposed work is to take advantage of photoswitchable fluorescent proteins to tag crucial polarity proteins that have been demonstrated to direct polarized cell behaviors. To this end, these studies will examine two of the most promising photoswitchable proteins, EosFP and Dronpa, under established live imaging conditions in the Drosophila embryo. After evaluating the behavior of EosFP and Dronpa in the fly embryo, transgenic lines carrying photoswitchable fusions to two critical epithelial polarity proteins will be established. This will allow, for the first time, the in vivo measurement of the turnover and dynamic localization of these polarity proteins. The approach used in this work is innovative because it will draw on cutting edge imaging techniques that are just beginning to be applied to Drosophila to establish the stability of the protein complexes that guide epithelial polarity. The significance of this work derives from the important functional conservation of these same polarity proteins in epithelial processes required for normal human development and homeostasis. PROJECT NARRATIVE: Epithelial tissues, such as the human skin and gut, maintain an essential polarity that allows the cells in these tissues to confront both the external environment of the organism and the aqueous internal environment. The maintenance of this polarity is essential to the barrier function of epithelia, and the loss of this polarity is associated with the metastasis of many epithelial cancers. The intention of this proposed study is to examine the dynamics by which polarity proteins are localized to specific places in the cell, thus leading to a better understanding of how the critical polarized function of epithelial tissues is maintained and can be subverted by disease.